Anna Marie Kenney

Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors

Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. We report that sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G1 cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors.

Sonic hedgehog Promotes G 1 Cyclin Expression and Sustained Cell Cycle Progression in Mammalian Neuronal Precursors

ABSTRACT Sonic hedgehog (Shh) signal transduction via the G-protein-coupled receptor, Smoothened, is required for proliferation of cerebellar granule neuron precursors (CGNPs) during development. Activating mutations in the Hedgehog pathway are also implicated in basal cell carcinoma and medulloblastoma, a tumor of the cerebellum in humans. However, Shh signaling interactions with cell cycle regulatory components in neural precursors are poorly understood, in part because appropriate immortalized cell lines are not available. We have utilized primary cultures from neonatal mouse cerebella in order to determine (i) whether Shh initiates or maintains cell cycle progression in CGNPs, (ii) if G1 regulation by Shh resembles that of classical mitogens, and (iii) whether individual D-type cyclins are essential components of Shh proliferative signaling in CGNPs. Our results indicate that Shh can drive continued cycling in immature, proliferating CGNPs. Shh treatment resulted in sustained activity of the G1 cyclin-Rb axis by regulating levels ofcyclinD1, cyclinD2, and cyclinEmRNA transcripts and proteins. Analysis of CGNPs fromcyclinD1−/− orcyclinD2 −/− mice demonstrates that the Shh proliferative pathway does not require unique functions ofcyclinD1 or cyclinD2 and that D-type cyclins overlap functionally in this regard. In contrast to many known mitogenic pathways, we show that Shh proliferative signaling is mitogen-activated protein kinase independent. Furthermore, protein synthesis is required for early effects on cyclin gene expression. Together, our results suggest that Shh proliferative signaling promotes synthesis of regulatory factor intermediates that upregulate or maintain cyclin gene expression and activity of the G1cyclin-Rb axis in proliferating granule neuron precursors.

Stabilization of N-Myc Is a Critical Function of Aurora A in Human Neuroblastoma

In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. In a shRNA screen of genes that are highly expressed in MYCN-amplified tumors, we have identified AURKA as a gene that is required for the growth of MYCN-amplified neuroblastoma cells but largely dispensable for cells lacking amplified MYCN. Aurora A has a critical function in regulating turnover of the N-Myc protein. Degradation of N-Myc requires sequential phosphorylation by cyclin B/Cdk1 and Gsk3. N-Myc is therefore degraded during mitosis in response to low levels of PI3-kinase activity. Aurora A interacts with both N-Myc and the SCF(Fbxw7) ubiquitin ligase that ubiquitinates N-Myc and counteracts degradation of N-Myc, thereby uncoupling N-Myc stability from growth factor-dependent signals.

The miR-17/92 Polycistron Is Up-regulated in Sonic Hedgehog–Driven Medulloblastomas and Induced by N-myc in Sonic Hedgehog–Treated Cerebellar Neural Precursors

Medulloblastoma is the most common malignant pediatric brain tumor, and mechanisms underlying its development are poorly understood. We identified recurrent amplification of the miR-17/92 polycistron proto-oncogene in 6% of pediatric medulloblastomas by high-resolution single-nucleotide polymorphism genotyping arrays and subsequent interphase fluorescence in situ hybridization on a human medulloblastoma tissue microarray. Profiling the expression of 427 mature microRNAs (miRNA) in a series of 90 primary human medulloblastomas revealed that components of the miR-17/92 polycistron are the most highly up-regulated miRNAs in medulloblastoma. Expression of miR-17/92 was highest in the subgroup of medulloblastomas associated with activation of the sonic hedgehog (Shh) signaling pathway compared with other subgroups of medulloblastoma. Medulloblastomas in which miR-17/92 was up-regulated also had elevated levels of MYC/MYCN expression. Consistent with its regulation by Shh, we observed that Shh treatment of primary cerebellar granule neuron precursors (CGNP), proposed cells of origin for the Shh-associated medulloblastomas, resulted in increased miR-17/92 expression. In CGNPs, the Shh effector N-myc, but not Gli1, induced miR-17/92 expression. Ectopic miR-17/92 expression in CGNPs synergized with exogenous Shh to increase proliferation and also enabled them to proliferate in the absence of Shh. We conclude that miR-17/92 is a positive effector of Shh-mediated proliferation and that aberrant expression/amplification of this miR confers a growth advantage to medulloblastomas.

Hedgehog and PI-3 kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors

Neuronal precursor cells in the developing cerebellum require activity of the sonic hedgehog (Shh) and phosphoinositide-3-kinase (PI3K) pathways for growth and survival. Synergy between the Shh and PI3K signaling pathways are implicated in the cerebellar tumor medulloblastoma. Here, we describe a mechanism through which these disparate signaling pathways cooperate to promote proliferation of cerebellar granule neuron precursors. Shh signaling drives expression of mRNA encoding the Nmyc1 oncoprotein (previously N-myc), which is essential for expansion of cerebellar granule neuron precursors. The PI3K pathway stabilizes Nmyc1 protein via inhibition of GSK3-dependent Nmyc1 phosphorylation and degradation. The effects of PI3K activity on Nmyc1 stabilization are mimicked by insulin-like growth factor, a PI3K agonist with roles in central nervous system precursor growth and tumorigenesis. These findings indicate that Shh and PI3K signaling pathways converge on N-Myc to regulate neuronal precursor cell cycle progression. Furthermore, they provide a rationale for therapeutic targeting of PI3K signaling in medulloblastoma.

Inhibition of phosphatidylinositol 3-kinase destabilizes mycn protein and blocks malignant progression in neuroblastoma

Amplification of MYCN occurs commonly in neuroblastoma. We report that phosphatidylinositol 3-kinase (PI3K) inhibition in murine neuroblastoma (driven by a tyrosine hydroxylase-MYCN transgene) led to decreased tumor mass and decreased levels of Mycn protein without affecting levels of MYCN mRNA. Consistent with these observations, PI3K inhibition in MYCN-amplified human neuroblastoma cell lines resulted in decreased levels of Mycn protein without affecting levels of MYCN mRNA and caused decreased proliferation and increased apoptosis. To clarify the importance of Mycn as a target of broad-spectrum PI3K inhibitors, we transduced wild-type N-myc and N-myc mutants lacking glycogen synthase kinase 3beta phosphorylation sites into human neuroblastoma cells with no endogenous expression of myc. In contrast to wild-type N-myc, the phosphorylation-defective mutant proteins were stabilized and were resistant to the antiproliferative effects of PI3K inhibition. Our results show the importance of Mycn as a therapeutic target in established tumors in vivo, offer a mechanistic rationale to test PI3K inhibitors in MYCN-amplified neuroblastoma, and represent a therapeutic approach applicable to a broad range of cancers in which transcription factors are stabilized through a PI3K-dependent mechanism.

Gli Activity Correlates with Tumor Grade in Platelet-Derived Growth Factor–Induced Gliomas

Gli signaling is critical for central nervous system development and is implicated in tumorigenesis. To monitor Gli signaling in gliomas in vivo, we created platelet-derived growth factor-induced gliomas in a Gli-luciferase reporter mouse. We find that Gli activation is found in gliomas and correlates with grade. In addition, we find that sonic hedgehog (SHH) is expressed in these tumors and also correlates with grade. We identify microvascular proliferation and pseudopalisades, elements that define high-grade gliomas as SHH-producing microenvironments. We describe two populations of SHH-producing stromal cells that reside in perivascular niche (PVN), namely low-cycling astrocytes and endothelial cells. Using the Ptc-LacZ knock-in mouse as a second Gli responsive reporter, we show beta-galactosidase activity in the PVN and in some tumors diffusely throughout the tumor. Lastly, we observe that SHH is similarly expressed in human gliomas and note that an intact tumor microenvironment or neurosphere conditions in vitro are required for Gli activity.

N-myc Is an Essential Downstream Effector of Shh Signaling during both Normal and Neoplastic Cerebellar Growth

We examined the genetic requirements for the Myc family of oncogenes in normal Sonic hedgehog (Shh)-mediated cerebellar granule neuronal precursor (GNP) expansion and in Shh pathway-induced medulloblastoma formation. In GNP-enriched cultures derived from N-myc(Fl/Fl) and c-myc(Fl/Fl) mice, disruption of N-myc, but not c-myc, inhibited the proliferative response to Shh. Conditional deletion of c-myc revealed that, although it is necessary for the general regulation of brain growth, it is less important for cerebellar development and GNP expansion than N-myc. In vivo analysis of compound mutants carrying the conditional N-myc null and the activated Smoothened (ND2:SmoA1) alleles showed, that although granule cells expressing the ND2:SmoA1 transgene are present in the N-myc null cerebellum, no hyperproliferation or tumor formation was detected. Taken together, these findings provide in vivo evidence that N-myc acts downstream of Shh/Smo signaling during GNP proliferation and that N-myc is required for medulloblastoma genesis even in the presence of constitutively active signaling from the Shh pathway.

N-myc Can Substitute for Insulin-Like Growth Factor Signaling in a Mouse Model of Sonic Hedgehog–Induced Medulloblastoma

Medulloblastoma is a malignant brain tumor that arises in the cerebellum in children, presumably from granule neuron precursors (GNP). Advances in patient treatment have been hindered by a paucity of animal models that accurately reflect the molecular pathogenesis of human tumors. Aberrant activation of the Sonic hedgehog (Shh) and insulin-like growth factor (IGF) pathways is associated with human medulloblastomas. Both pathways are essential regulators of GNP proliferation during cerebellar development. In cultured GNPs, IGF signaling stabilizes the oncogenic transcription factor N-myc by inhibiting glycogen synthase kinase 3beta-dependent phosphorylation and consequent degradation of N-myc. However, determinants of Shh and IGF tumorigenicity in vivo remain unknown. Here we report a high frequency of medulloblastoma formation in mice following postnatal overexpression of Shh in cooperation with N-myc. Overexpression of N-myc, alone or in combination with IGF signaling mediators or with the Shh target Gli1, did not cause tumors. Thus, Shh has transforming functions in addition to induction of N-myc and Gli1. This tumor model will be useful for testing novel medulloblastoma therapies and providing insight into mechanisms of hedgehog-mediated transformation.

Neural Precursor Cycling at Sonic Speed: N-Myc Pedals, GSK-3 Brakes

Signaling by the sonic hedgehog (Shh) pathway is essential for neural precursor population expansion during normal central nervous system (CNS) development, and is implicated in the childhood brain tumor, medulloblastoma. The proto-oncogene N-myc plays essential roles as a downstream effector of Shh proliferative effects in neural precursors of the cerebellum, where medulloblastomas arise. It is likely that N-Myc has analogous functions in medulloblastomas and other CNS tumors where it is highly expressed due to altered regulation or gene amplification. Myc destabilization occurs in response to phosphorylation by GSK-3β. N-Myc degradation is required for cerebellar neural precursors to exit the cell cycle. During mitosis in cerebellar neural precursors, levels of N-Myc primed for phosphorylation by GSK-3β increase, due to cdk1 complex activity towards N-Myc. GSK-3β is kept in check by insulin-like growth factor signaling, which also plays critical roles in brain development and cancer. These findings indicate that therapeutic strategies targeting N-myc and the IGF pathway might be effective against medulloblastoma.